The goal of this project is to understand respiratory burst- independent microbial killing by human neutrophils at the molecular level. A potent, rapidly acting, broad-spectrum antimicrobial protein in human neutrophils has recently been characterized, partially purified and localized to the membrane of the azurophil (primary) granule. This material is designated azurophil-derived bactericidal factor, or ADBF. Initially, we will purify ADBF from azurophil granules, determine if it consists of multiple species or if it is a single entity and characterize its physicochemical properties with respect to other neutrophil-derived bactericidal proteins previously described. We will determine the subcellular location of ADBF in resting and phagocytizing neutrophils, using immunoelectron microscopy experiments, with special attention to whether ADBF is delivered to phagolysosomes containing ingested microorganisms. We will study the mechanism of action of ADBF by examining the interaction of purified 125I-labelled ADBF with the E. coli cell surface and by measuring the effect of purified ADBF on E. coli outer membrane, cytoplasmic membrane and intracellular synthetic machinery. We will isolate E. coli mutants resistant to each ADBF species identified and to combinations of ADBF species. These mutants will be used to evaluate the contribution of ADBF to oxygen-dependent and independent microbial killing by human neutrophils. Such mutants may also provide additional information concerning the mode of action of ADBF.